A Panel of VHH Antibodies Against Sabin Type 1 Poliovirus D-Antigen Reveals Strain-Specific and Cross-Serotype Reactivity

Figures

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  Figure 1. Schematic representation of the construction and selection of a phage library of VHH antibodies against poliovirus Sabin type 1. Created in BioRender. Shishova, A. (2026) https://BioRender.com/hboouqd.

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  Figure 2. Analytical gel electrophoresis of colony PCR products, M—DNA size marker (1kb, Evrogen, Russia): (A) after primary transformation of the phagemid library; (B) after the third round of selection.

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  Figure 3. Pools of VHH antibodies selected against poliovirus Sabin strain type 1 during three rounds of biopanning were analyzed for binding to the target antigen—the purified D-antigen of poliovirus Sabin type 1—as well as to antigens of the other two serotypes (types 2 and 3) to assess cross-reactivity. The optical density (OD450) of each sample was normalized to the background value (0.055), and the signal-to-background ratio was calculated. Data are shown as individual values for each clone. The selection threshold was set at a signal-to-background ratio ≥ 40 for binding to the target antigen; clones with a ratio above this value and with no cross-reactivity to the other serotypes were selected for further analysis. Each circle corresponds to individual clone signal.

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  Figure 4. Multiple sequence alignment of recombinant VHH clone amino acid sequences visualized using CLC Sequence Viewer (QIAGEN, Germantown, MD, USA).

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  Figure 5. SDS–PAGE analysis of purified VHH antibodies (20% gel) under denaturing conditions. The expected molecular weight of the target protein is ~20 kDa.

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  Figure 6. (A) Specificity of recombinant VHH antibodies against Sabin type 1 poliovirus as determined by ELISA. The figure shows OD450 values as a function of antigen concentration (DU/mL) for monoclonal antibodies S1-C7, S1-D1, S1-D7, S1-D9, S1-E8, and S1-G5. Legend: solid blue line—D-antigen of Sabin type 1; solid green line—D-antigen of Sabin type 2; solid black line—D-antigen of Sabin type 3; dashed blue line—inactivated poliovirus, Mahoney strain; dashed green line—inactivated poliovirus, MEF-1 strain; dashed black line—inactivated poliovirus, Saukett strain. ELISA results were visualized using GraphPad Prism 8.2.1. software,. (B) Schematic representation of the ELISA used to determine the specificity of the obtained VHH antibodies against Sabin type 1 poliovirus. Recombinant VHH antibodies were tested for reactivity with D-antigens of Sabin poliovirus strains (types 1, 2, and 3) and wild-type strains (Mahoney, MEF-1, and Saukett), with detection using polyclonal antibodies specific for the corresponding serotypes. Results are presented as means ± standard deviation from three independent experiments (n = 3). Asterisks indicate statistical significance calculated using two-way ANOVA.…

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  Figure 7. (A) Specificity of monoclonal antibody S1-C7 and polyclonal antibodies raised against the Sabin type 1 strain for D- and H-antigens of type 1 polioviruses (Sabin and Mahoney) as determined by ELISA. ELISA scheme with immobilized monoclonal VHH antibodies; (B) ELISA scheme with immobilized polyclonal antibodies. Subsequent steps are identical for both panels: addition of antigen (native D-antigen or H-antigen obtained by heat treatment), detection with biotinylated polyclonal antibodies against poliovirus, HRP-conjugated streptavidin, and TMB substrate. The upper graphs show changes in optical density (OD450) as a function of poliovirus antigen concentration (DU/mL). The solid line represents the D-antigen, and the dashed line the H-antigen. Blue lines indicate the Sabin strain; black lines indicate the Mahoney strain. (A) VHH antibody S1-C7; (B) polyclonal serum. Results are presented as means ± standard deviation from three independent experiments (n = 3). Asterisks indicate statistical significance calculated using two-way ANOVA. Schematic representation of the ELISA created using BioRender.com. Shishova, A. (2026) https://BioRender.com/9q6mrsb.

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  Figure 8. Reactivity of the S1-C7 antibody toward Mahoney-derived virus-like particles (VLPs) in sandwich ELISA. Detection was performed using biotinylated polyclonal antibodies against Sabin type 1 poliovirus. Lines: solid blue—Sabin type 1 D-antigen; solid black—Mahoney D-antigen; dashed black—VLPs.